Optimal Combination of Glycine, Asparagine, and Phenylalanine Promotes α-Casein Synthesis and Secretion in MAC-T Cells Through Activation of the PI3K-AKT-mTOR Pathway

Efficient milk protein synthesis in dairy cows, particularly casein production, is crucial for milk quality but has low nitrogen conversion efficiency. This study aimed to determine whether an optimal ratio of glycine, asparagine, and phenylalanine could synergistically promote α-casein synthesis in bovine mammary epithelial cells (MAC-T) and to elucidate its mechanism via the PI3K-AKT-mTOR signaling pathway. Single-factor experiments and response surface central composite design were conducted to determine the optimal amino acid combination. α-Casein synthesis was measured by ELISA, gene expression by RT-qPCR, and protein phosphorylation by Western blot. A PI3K-specific inhibitor (LY294002) was used in a blocking experiment to validate the involvement of the PI3K-AKT-mTOR pathway. Results: The optimal ratio was 9.898 mmol/L glycine, 7.014 mmol/L asparagine, and 5.865 mmol/L phenylalanine (molar ratio 1.69:1.20:1.00). This combination significantly increased α-casein synthesis and secretion compared to any single amino acid (p < 0.01), demonstrating a synergistic effect. It also upregulated CSN1S1 and CSN1S2 expression and activated the PI3K-AKT-mTOR pathway at both transcriptional and translational levels. The addition of LY294002 completely abolished these effects, confirming the pathway’s crucial role. The optimal combination of glycine, asparagine, and phenylalanine synergistically enhances α-casein synthesis in MAC-T cells by activating the PI3K-AKT-mTOR pathway. These findings provide a theoretical basis for developing targeted amino acid supplementation strategies to improve milk protein production in dairy cows.